The present invention relates to a colorimetric method for effecting a quantitative analysis for specific substances contained in a sample and an apparatus for carrying out the colormetric method.
Such a colorimetric method has been widely used in the biochemistry field, etc. In a conventional colorimetric apparatus, it is necessary to perform a 100% adjustment for a transmitted light flux (zero adjustment of an absorbance) by using water, etc. In the case of performing a photometric measurement with the aid of a reagent having extremely small absorbance, there occurs no problem due to this 100% adjustment, but in the case of using a reagent having high absorbance, measuring results might be largely affected by the absorbance of the reagent. Therefore, recently, the 100% adjustment has been generally effected by using the reagent to be used in measurement.
GOT (Glumatate Oxalacetate Transaminase) and GPT (Glumatate Pyruvate Transaminase) contained in a blood sample have been measured in an ultraviolet colorimetric (performing the photometric measurement by using ultraviolet light). In this ultraviolet colorimetric measurement, the absorbance is decreased in accordance with the reaction of GOT or GPT with the reagent, and thus when the 100% adjustment, i.e. zero absorption adjustment is performed by using the reagent, it is not possible to effect the colorimetric measurement, because the absorbance decreases below zero. Therefore, in this case, the photometric measurement is performed by deriving a difference between the transmitted light flux of the reagent and that after a sample reaction. However, since this method uses the reagent having the high absorbance, it is not possible to effect an accurate photometric measurement. This reason will be explained hereinafter.
In FIG. 1, among various test-items showing a general increasing tendency of the absorbance, a curve A shows an absorbance variation of a glucose which is selected as a type using a low absorbance reagent, and a curve B shows the absorbance variation of a magnesium which is a typical item using a high absorbance reagent. Here, both of them are measured by using a light flux having 500 nm wavelength. Table 1 shows a transmissivity and the absorbance, in the case of taking a measurement of only the reagent or after the reaction between the sample and the reagent.
TABLE 1 ______________________________________ reagent after reaction ______________________________________ glucose transmissivity 89.1% 14.1% absorbance 0.05 0.85 magnesium transmittivity 8.96% 6.31% absorbance 1.05 1.20 ______________________________________
In Table 1, the absorbance is calculated by a formula log I.sub.0 /I, where I.sub.0 is the intensity of an incident light flux and I is that of a transmitted light flux. Therefore, for example, in the case of performing the photometric measurement only for the reagent used in measuring the glucose, since the incident light is 100% and the transmitted light is 89.1%, the absorbance of this reagent is obtained from the calculation log 100/89.1.congruent.0.05.
After reaction between the glucose and the reagent, the transmissivity is decreased to 14.1% and the absorbance is increased to 0.85. In this case, if it is assumed that the transmissivity is varied by 0.1%, i.e. the transmissivity becomes 14.2%, the absorbance becomes log 100/14.2=0.847 and thus an amount of the absorbance deviation becomes 0.85-0.847=0.003. Contrary to this, in the magnesium measurement, when the transmissivity is varied by the same amount of 0.1% and is changed from 6.31% to 6.41%, the absorbance becomes log 100/6.41.congruent.1.193 and thus the amount of the absorbance deviation becomes 0.007. Therefore, the amount of the absorbance deviation in the magnesium measurement is larger than that in the glucose measurement.
Moreover, in the case of performing the photometry for the reagent, if it is assumed that the transmissivity is varied by 0.1%, the amount of the absorbance deviations in the glucose measurement and the magnesium measurement become 0.0005 and 0.005, respectively. Therefore, in this case, the amount of the absorbance deviation in the magnesium measurement is larger by 10 times than that of the glucose measurement. In the case of performing the photometry by using the high absorbance reagent, slight variation and error in the amount of transmitted light affect largely the absorbance measurement as compared with the case using the low absorbance reagent, even if the 100% adjustment is performed by using the reagent. Therefore, an accuracy of the result is varied in accordance with the test-items.
Contrary to this, in the case of performing the ultra-violet colorimetric measurement for the GOT or the GPT, the absorbance after reaction between the sample including the GOT or the GPT and the reagent having the high absorbance such as 1.0-1.5 is decreased by 0.2-0.3 as compared with that for only reagent. Moreover, in an extreme case, the decreased amount of the absorbance after reaction becomes 0.7-0.8. If the absorbance is assumed to be a, an equation I=I.sub.0 /10.sup.a is derived from an equation log I.sub.0 /I=a. Therefore, if the absorbance is decreased by 0.3, the amount of the transmitted light flux is increased by 1/10.sup.-0.3 (=10.sup.0.3) times, i.e. about two times. In the same manner, if the absorbance is decreased by 0.5 and 0.7, respectively, the amount of transmitted light is increased by about three (=10.sup.0.5) and five (=10.sup.0.7) times, respectively. Since the larger the amount of transmitted light becomes, the better an S/N ratio becomes, the accuracy of the photometric measurement is improved. However, if the deviation of the amount of transmitted light becomes large as mentioned above, the S/N ratio, i.e. the accuracy, is varied correspondingly, and thus the accuracy of the colorimetric measurement cannot be stably maintained.